One means for improving a plant includes a “transformation technique”, whereby a desired recombinant gene for modifying a character is introduced into the plant. Efficient and quick transformation techniques are extremely important for the molecular breeding of useful plants, in particular grain crops, which are important staple foods.
A majority of grain crops (e.g., rice, wheat, barley, and corn) are classified as monocotyledons. Various transformation techniques for transforming monocotyledons have hitherto been developed. Transformation techniques are generally classified into direct transformation techniques and indirect transformation techniques.
Examples of direct transformation techniques include electroporation techniques (see Non-Patent Documents 1 and 2), particle gun techniques (see Non-Patent Document 3) and polyethylene glycol (PEG) techniques (see Non-Patent Document 4). Electroporation techniques and particle gun techniques have been generally used as methods for transforming monocotyledons which can achieve relatively efficient gene introduction.
An example of an indirect transformation technique is an Agrobacterium-mediated transformation technique (hereinafter, this may also be referred to as an “Agrobacterium transformation technique”). Agrobacteria are a species of plant pathogenic bacteria. Agrobacteria are characterized in that, when a plant is infected therewith, a T-DNA region which is present on the plasmids that Agrobacteria have (e.g., Ti or Ri plasmid) is incorporated into the plant. The Agrobacterium transformation technique utilizes the incorporation of the T-DNA region into plants as a means for introducing genes into plants. In short, a plant is infected with an Agrobacterium which contains a desired recombinant gene. After infection, the desired recombinant gene is transferred from the Agrobacterium into plant cells so as to be incorporated into the plant genome.
The Agrobacterium transformation technique is sufficiently established so far as dicotyledons are concerned. A large number of stable transformed plants have already been created which express desired recombinant genes.
On the contrary, it has conventionally been recognized as difficult to apply the Agrobacterium transformation technique to monocotyledons. For example, Portrykus et al. (Non-Patent Document 5) report that monocotyledons cannot be infected with Agrobacteria. On the other hand, a great deal of attempts have been made to transform monocotyledons by using Agrobacteria, which have shed light on the possibility of applying the Agrobacterium transformation technique to monocotyledons.
For example, Raineri et al. took the scutellum of rice, scarred it, and placed it on a medium which induces dedifferentiation; a few days later, the scutellum portion was infected with an Agrobacterium. As a result, although normally redifferentiated plant bodies were not obtained, calluses having a foreign gene introduced therein were successfully induced (see Non-Patent Document 6).
Japanese Patent No. 2649287 (Patent Document 1) discloses an Agrobacterium transformation technique for rice and corn. According to this method, it is necessary to employ cultured tissue (e.g., calluses), which are dedifferentiated, as a plant sample to be transformed by an Agrobacterium. Therefore, prior to infection with an Agrobacterium, it generally takes 3 to 4 weeks to induce dedifferentiation in order to produce dedifferentiated culture tissue from a plant sample to be transformed (e.g., a leaf section).
Japanese Patent No. 3141084 (Patent Document 2) discloses a transformation method for monocotyledons using germinated seeds which are germinated by being subjected to pre-culture with a medium containing 2,4-D for 4-5 days after sowing. When using the transformation method disclosed in Patent Document 2, monocotyledons can be transformed more quickly as compared to the transformation method described in Patent Document 1, and therefore, it is excellent. However, it was considered that, prior to infection with an Agrobacterium, pre-culture for about 4-5 days would still be required.
As described above, for the transformation of monocotyledons via an Agrobacterium according to conventional techniques, a long period of time is required to prepare plant tissue/plant cells, which can be infected with an Agrobacterium (e.g., callusing of monocotyledon cells). The time required for the aforementioned preparation has been a barrier to promoting the efficiency of molecular breeding of monocotyledon cells. Therefore, in order to build up molecular breeding, it is significantly important to reduce the time required for transformation.
Therefore, it is desired that a method for transforming plant cells more quickly than the conventional techniques be established.
Patent Document 1: Japanese Patent. No. 2649287
Patent Document 2: Japanese Patent No. 3141084
Non-Patent Document 1: Shimamoto K. et al., Nature, 338: 274-276, 1989
Non-Patent Document 2: Rhodes C. A. et al., Science, 240: 204-207, 1989
Non-Patent Document 3: Christou P. et al., Bio/Technology 9: 957-962, 1991
Non-Patent Document 4: Datta, S. K. et al., Bio/Technology, 8: 736-740, 1990
Non-Patent Document 5: Portrykus et al., BIO/TECHNOLOGY, 535-542, 1990
Non-Patent Document 6: Raineri, D. M. et al., Bio/Technology, 8: 33-38, 1990